Fig. 2. HF and Febrifugine Inhibit Prolyl tRNA Synthetase Activity In Vitro.
A) Rabbit reticulocyte lysate (RRL) was incubated with luciferase mRNA and translation quantitated in a luminescence assay. A solution of a mixture of amino acids or individual amino acids was added to give 1 mM of each in the reaction to rescue translational inhibition. Mix1: Asn, Arg, Val, Glu, Gly; Mix2: Lys, Ile, Tyr, Asp, Trp; Mix 3: His, Met, Leu, Ala, Thr; Mix 4: Ser, Phe, Pro, Gln. Note log scale of y-axis. B) Effect of HF and its derivatives in the presence or absence of proline supplementation on translation were assayed as in A. Error bars reflect standard deviation of triplicate determinations. C) Short myc-tagged polypeptides of identical sequence (see Supplementary Methods) with the exception that NoPropep lacks proline, while Propep contains a proline dipeptide, were translated in RRL in the presence of indicated inhibitors (each at a concentration of 1 μM, proline added at 1 mM). Translation was examined by anti-myc Western blot. D) 14C Pro or 35S Met (Perkin-Elmer) were incubated with RRL (Promega) and 1 μg/μl total bovine tRNA (Sigma) in the presence or absence of HF or MAZ1310 for 10′, tRNA was isolated using a MirVana tRNA isolation kit (Ambion), and radioactivity in tRNA was measured by liquid scintillation counting. Error bars reflect standard deviation of triplicate determinations.